The long term goal of this proposal is to elucidate the mechanisms controlling fibroblast activation. These mechanisms are relevant for wound healing as fibroblast activation has been suggested to be a rate-limiting step in the progression of skin repair and the disregulation of this state is associated to impaire healing. Moreover, a detailed understanding of the molecular components of these mechanisms will provide tools to modify the course of wound healing. Interference with the fibronectin receptor-mediated cell adhesion is sufficient to activate NFkappaB and to establish an IL-1a autocrine loop necessary for the induction of the matrix metalloproteinase collagenase 1/MMP-1 (CL-1) and stromelysin/MMP-3. This signal transduction pathway proceeds through the activation of the GTPase Rac and changes in mitochondrial function, which include membrane potential loss, to produce radical oxygen species (ROS). The specific objective of this proposal is to determine how Rac, ROS production and mitochondrial membrane potential are mechanistically related. 1. To determine the mechanism eliciting ROS in the respiratory chain upon integrin engagement. Substrate-dependent oxygen consumption concomitant with H2O2 production will be measured in simulated cells to establish the role of the different respiratory complexes in this signaling mechanism. It is expected that these experiments will evidence changes in the function of one of the respiratory chain complexes. 2. To determine the mechanism for mitochondrial potential loss and the role of this event during integrin signaling. The role of the permeability transition pore in controlling membrane potential will be evaluated using pharmacological inhibitors. These studies will be complemented by evaluating the role of mitochondrial uncoupling in signal transduction, through expression of uncoupling protein 3 (UCP3). 3. To determine whether Rac regulates mitochondrial function to modify MMP production. Mitochondrial respiratory chain activity and membrane potential will be evaluated upon adenoviral expression of the constitutively active mutant of Rac.